Biomass is a very promising resource for replacing fossil raw materials in applications in which carbon is indispensable, such as liquid fuels, materials and chemicals. However, current technologies are primarily based on the fermentation of sugars derived from starch and sugar crops and thus raise concerns about the diversion of farmlands or crops to biofuels production in detriment of the food supply.
Biorefinery offers the potential to use a wide variety of non-food biomass resources such as agricultural residues, forestry and municipal wastes or dedicated crops such as switchgrass or miscanthus, to produce valuable biochemicals, biomaterials and biofuels. Production from this lignocellulosic biomass is thus an attractive alternative that does not interfere with food security.
Biorefinery is based on the conversion of lignocellulosic biomass into monomeric sugars that can be chemically transformed or fermented into various compounds such as biofuels (e.g. bio-ethanol, butanol) or biochemicals (e.g. plastics, detergents, vitamines).
However, one barrier to biorefinery is that the sugars are trapped inside the lignocellulose composed of cellulose, hemicellulose and lignin. Consequently, lignocellulosic biomass has to be pretreated to break down the shield formed by lignin and hemicellulose and disrupt the crystalline structure of cellulose to access the polymer chains. This pretreatment may include physical, chemical and/or biological methods. Cellulose and hemicellulose polymers are then enzymatically or chemically hydrolyzed into monomeric sugars.
Enzymatic hydrolysis of these polymers may be carried out by cellulolytic and hemicellulolytic enzymes. In particular, the enzymatic degradation of cellulose involves synergistic actions of three major classes of enzymes endoglucanases (EG, EC 3.2.1.4), cellobiohydrolases (CBH, EC 3.2.1.91) and beta-glucosidases (BG, EC 3.2.1.21). Indeed, cellobiohydrolases processively cleave cellulose chains at the ends to release soluble cellobiose or glucose, endoglucanases randomly hydrolyze accessible intramolecular β-1,4-glycosidic bonds of cellulose chains to produce new chain ends and beta-glucosidases hydrolyze cellobiose to glucose. Since hemicellulose comprised different sugar monomers such as xylose, mannose or arabinose, the hemicellulytic enzymes are more complex and may involve, for instance, xylanases and mannases.
Currently, many important commercial enzyme preparations for biomass conversion of cellulose or hemicellulose are from the fungus Trichoderma reesei (also named Hypocrea jecorina) that secretes, for instance, cellobiohydrolases (CBH I and CBH II) and several endoglucanases (e.g. EGI, EGII or EGIII) and beta-glucosidases (e.g. BG I).
Enzymatic hydrolysis and fermentation may be conducted separately. This process is termed process SHF for “Separate enzymatic hydrolysis and fermentation”. In this case, pretreated lignocellulosic biomass is enzymatically hydrolysed to monomeric sugars and subsequently fermented in separate units. The advantage of SHF is the ability to carry out each step under optimal conditions, i.e. enzymatic hydrolysis at 45-50° C. and fermentation at about 30° C.
Enzymatic hydrolysis and fermentation may also be conducted simultaneously in the same bioreactor. This process is termed process SSF for “Simultaneous saccharification and fermentation”. In this case, sugars produced by the hydrolysing enzymes are consumed immediately by the fermenting microorganism present in the culture. This process is preferred with respect to process integration and simplification but it is less efficient than SHF. Indeed, the main problem for SSF process is that optimum conditions for the enzymatic hydrolysis and fermentation have to be as close as possible. However, the optimum temperatures and pH of cellulases are usually reported to be in the range of 45 to 55° C. and pH 4 to 5 and the optimum temperatures and pH for the most frequently used microorganism for fermenting ethanol in industrial process, i.e. Saccharomyces cerevisiae, are about 30° C. and pH 6. The SSF is thus usually performed at about 38° C., which is a compromise between the optimal conditions for hydrolysis and fermentation. However, this made the SSF process much slower (Hari Krishna et al., 2000; Saha et al., 2005).
In order to improve SSF and increase ethanol productivity, thermotolerant yeasts have been developed such as Kluyveromyces marxianus (Ballesteros et al., 2004) or Candida acidothermophilum (Kadam et al., 1997). Because degradation of cellulose at elevated temperature provides many benefits, such as increased cellulase activity, less energy cost for cooling, and decreased risk of contamination, thermostable cellulases have also been developed and may be advantageously used in combination with these thermotolerant yeasts (Hong et al., 2007). As illustration, thermostable T. reesei endoglucanase I variants having substitution at position 230, 113 or 115 of the mature protein, have been disclosed in the patent application WO 2012/036810.
In an alternative approach, enzymatic hydrolysis and fermentation may be carried out by a single community of microorganisms. This process is termed process CBP for “Consolidated Bioprocessing”. For this process, ethanol producers can be modified to become cellulase producers or vice-versa. For example, a strain of Saccharomyces was modified to express endoglucanase and beta glucosidase (Den Haan et al., 2007). This strain was thus able to grow on cellulose and to converts cellulose to ethanol in one step. However, to date, yields of this process remains insufficient.
Thus, even if much valuable work has been performed during recent years, improvements remain necessary to make biorefinery, and in particular bio fuel production process, economically feasible. In particular, it would be of great interest to increase the productivity and reduce the production cost of processes wherein saccharification and fermentation are conducted simultaneously.